Peptides and polypeptides derived from the submaxillary gland of the rat, corresponding polyclonal and monoclonal antibodies, corresponding hybridomas and uses of these products for diagnosis, for detection or therapeutic purposes

ABSTRACT

The invention relates to peptides having the formula (I): X-His-Asn-Pro-Y wherein X is a rest Gln or pyro-Glu. Y is a group OH or a rest of basic aminoacid.

The present invention relates to novel peptides which are maturationproducts of polypeptides secreted by the submaxillary gland of the ratand analogues of these peptides.

Many polypeptides having defined biological properties are synthesizedin large quantities in the submaxillary gland (SMG) of rodents, and inparticular in the SMG of the mouse. These proteins, comprising the nervegrowth factor (NGF), the epidermal growth factor (EGF) and renin, have acertain number of properties in common. They are all synthesized in thesame type of cell, namely the cells of the convoluted tubules GCT(granular convoluted tubular), in response to various hormonal stimuli,in particular to androgens. Furthermore, it is possible to observe thepresence of these secretory proteins in the saliva of the mouse, andthey are synthesized in the form of precursors which become active aftermaturation processes which may involve proteases of the kallikrein type.Some of these proteases of the kallikrein type are also synthesized inthe SMG under the control of androgens.

Attempts at the characterization of the genes controlled by theandrogens in the SMG of the rat have led the inventors to analyse theelectrophoretic profile of the in vitro translation products of the mRNAof this tissue.

A specific mRNA of the submaxillary gland of the male rat has beenisolated. This mRNA corresponds to a polypeptide which has beendesignated SMR1. This polypeptide gives maturation products withphysiological activity. The present invention relates mainly to thesepeptides and to other peptides having similar properties.

Thus the subject of the present invention is peptides of formula:X-His-Asn-Pro-Y  Iin which:

-   -   X represents a glutamine (Gin) or pyroglutamic acid (pyro-Glu)        residue,    -   Y represents a OH group or a residue of a basic amino acid.

The basic amino acids may be lysine or arginine.

More specifically, the subject of the present invention is peptides offormula: Gln-His-Asn-Pro II pyro-Glu-His-Asn-Pro III Gln-His-Asn-Pro-ArgIV pyro-Glu-His-Asn-Pro-Arg V Gln-His-Asn-Pro-Lys VIpyro-Glu-His-Asn-Pro-Lys VII

Another subject of the present invention is the polypeptide SMR1 whichgives the maturation products of formulae II, III, IV and V. Thispolypeptide corresponds to the formula: 1 Met Lys Ser Leu Tyr Leu IlePhe Gly Leu Trp Ile                             20 Leu Leu Ala Cys PheGln Ser Gly Glu Gly Val Arg                     30 Gly Pro Arg Arg GlnHis Asn Pro Arg Arg Gln Gln             40 Asp Pro Ser Thr Leu Pro HisTyr Leu Gly Leu Gln     50                                      60 ProAsp Pro Asn Gly Gly Gln Ile Gly Val Thr Ile                                    70 Thr Ile Pro Leu Asn Leu Gln ProPro Arg Val Leu                             80 Val Asn Leu Pro Gly PheIle Thr Gly Pro Pro Leu                 90 Val Val Gln Gly Thr Thr GluTyr Gln Tyr Gln Trp             100 Gln Leu Thr Ala Pro Asp Pro Thr ProLeu Ser Asn     110                                     120 Pro Pro ThrGln Leu His Ser Thr Glu Gln Ala Asn                                    130 Thr Lys Thr Asp Ala Lys Ile SerAsn Thr Thr Ala                             140 Thr Thr Gln Asn Ser ThrAsp Ile Phe Glu Gly Gly Gly Lys

Another subject of the present invention is monoclonal and polyclonalantibodies directed against the peptides and the polypeptide accordingto the invention.

Another subject of the present invention is hybridomas producingmonoclonal antibodies directed against the peptides I to VII and thepolypeptide according to the invention.

Another subject of the present invention is a procedure for the assay ordetection of the peptides and polypeptides according to the invention intissues and biological fluids which comprises the utilization ofmonoclonal or polyclonal antibodies according to the invention.

For this purpose, it is possible to utilize in particular a method ofthe RIA type using a peptide labelled by a radioisotope, and thecompetition between this peptide and the peptide to be assayed(Niswender G. D. et al; Proc. Soc. Exp. Biol. 128, 807, 1968). It isalso possible to utilize a method of the ELISA type using, for example,a peptide bound to a support and the competition between this peptideand the peptide to be assayed for antibodies prepared against thispeptide. The antibodies retained by the peptide bound to the support aredetected by antibodies directed against the former and linked to anenzyme (method derived from Avromeas J. and Guilbert B, C.R. Acad. Sci.Paris 1971, 273, 2305).

The peptides according to the present invention may be prepared in astandard manner by peptide synthesis in liquid or solid phase bysuccessive couplings of the different amino acid residues to beincorporated (from the N-terminal end toward the C-terminal end inliquid phase, or from the C-terminal end to the N-terminal end in solidphase) and the N-terminal ends and the reactive side chains of which areblocked beforehand by standard groupings.

For this synthesis on solid phase it is possible to utilize inparticular the technique described by Merrifield, in the articleentitled “Solid phase peptide synthesis” (J. Am. Chem. Soc., 85,2149-2154).

In order to produce a peptide chain according to the Merrifieldprocedure, recourse is had to a very porous polymeric resin, to whichthe first C-terminal amino acid of the chain is bound. This amino acidis bound to the resin through the intermediary of its carboxyl group andits amino function is protected, for example by the t-butoxycarbonylgroup.

When the first C-terminal amino acid is thus bound to the resin, theprotecting group is removed from the amine function by washing the resinwith an acid. In the case in which the protecting group for the aminefunction is the t-butoxycarbonyl group, it may be removed by treatmentof the resin with the aid of trifluoroacetic acid.

The second amino acid, which provides the second residue of the desiredsequence, is then coupled to the deprotected amine function of the firstC-terminal amino acid bound to the chain. Preferably, the carboxylfunction of this second amino acid is activated, for example, by meansof dicyclohexylcarbodiimide, and the amine function is protected, forexample, by the t-butoxycarbonyl.

In this way, the first part of the desired peptide chain is obtainedwhich contains two amino acids and the terminal amine function of whichis protected. As previously, the amine function is deprotected and it isthen possible to proceed to the attachment of the third residue undersimilar conditions to those for the addition of the second C-terminalamino acid.

In this manner, the amino acids which will constitute the peptide chainare attached one after the other to the amine group, deprotectedbeforehand each time, of the portion of the peptide chain already formedand which is attached to the resin.

When the whole of the desired peptide chain is formed, the protectinggroups are removed from the different amino acids constituting thepeptide chain and the peptide is cleaved from the resin, for example,with the aid of hydrogen fluoride.

The peptide thus obtained can be purified, for example by means ofcolumn chromatography.

The SMR1 peptide or derivatives of this peptide can also be obtainedwith the aid of the techniques of genetic engineering; they can also beobtained by purification from biological material by means of techniquesof chromatography or precipitation similar to those used, for example,for the purification of human growth hormone starting from thehypophysis.

The monoclonal and polyclonal sera can be prepared according to astandard technique. For this purpose the tetrapeptides or thepentapeptides or multimeric derivatives of these peptides can be coupledto immunogenic agents such as KLH (Keyhole Lympet Hemocyanin),ovalbumin, bovine serumalbumin etc, by a coupling agent such asglutaraldehyde. The SMR1 protein and the derivatives of this protein(peptides derived from this protein, or hybrid proteins containing apart or all of SMR1 linked to another protein such as protein A) canalso be injected directly.

The immunizations can be performed in a standard manner, for example, inthe rabbit and the mouse by injecting into the animal 100 micrograms,for example, of the coupling product in the presence of Freund'sadjuvant 3 to 4 times at intervals of 3 weeks. It is thus possible toobtain polyclonal sera in the rabbit.

The hybridomas and the monoclonal antibodies can be obtained by means ofthe standard procedures.

The isolation of the mRNA, which corresponds to the SMR1 polypeptide,and the properties of the polypeptide and the maturation products willbe described in more detail below.

1) Materials and Methods

Animals and Hormonal Treatments

10 weeks old male and female Wistar rats were obtained from Iffa-Credo.The androgens were removed by castration and 10 days later 35 mg oftestosterone (Sterandryl retard, Roussel) were injected by theintraperitoneal route in the cases indicated. In the cases indicated,the same dose of testosterone was administered to female rats. 8 weeksold DBA/2 and “Swiss” mice were obtained from the Pasteur Institute.

Extraction of the RNA and In Vitro Translation

The RNA was prepared from rat and mouse tissues as described in theliterature (Tronik D. et al. 1987, EMBO J. 6, 983-987). The in vitrotranslation of the RNAs was carried out by using the mRNA-dependenttranslation system as a lysate in the presence of mRNA-dependentreticulocytes (Pelham H. RB et al., 1976, Eur. J. Biochem. 67, 247-256).The products were analysed by electrophoresis on a denaturingpolyacrylamide-NaDodSO₄ gel.

Cloning and Characterization of the cDNA Coding for SMR1

Poly(A) RNA obtained from SMG of male Wistar-rats was used as matrix forthe reverse transcriptase, and the double-stranded cDNAs were obtainedby means of the DNA synthesis system of Amersham, with the protocolsupplied by the manufacturer. The double-stranded cDNA was then insertedinto the PstI site of pUC9 by the method of the oligo-d(C) ends(Maniatis, T. et al. (1982) in Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) pp 241-242).The host bacteria (bacterial strain DH 5-1 derived from DH1 and yieldinghigh efficiencies of transformation, see Hanatian DNA cloning vol. 1, p.111) were transformed and the colonies were screened by hybridizationwith the probes described below. In brief, the mRNA derived from the SMGof males and females was fractionated in a 5-20% sucrose gradient. Thefractions enriched in mRNA of low molecular weight (which were shown byin vitro translation to contain the mRNA coding for SMR1) wereprecipitated with ethanol, and they were used as matrix for the reversetranscriptase in the presence of dGTP and dCTP radiolabelled with ³²P.About 3000 recombinants were screened on filters in duplicate sampleswith the radiolabelled cDNA obtained from the SMG of male and femalerats. The clones selected demonstrated a strong hybridization with themale cDNA probe but a very weak hybridization with the female probe. Therecombinant clones were identified by experiments of inhibition oftranslation of the mRNAs in a cell-free system by DNA-mRNA hybridization(Paterson et al. (1977) Proc. Natl. Acad. Sci. USA, 74, 4370-4374).

Sequencing of the cDNA Coding for SMR1

With the aid of various restriction enzymes, the cDNA coding for SMR1was cut and the fragments obtained were subcloned in the vector M13 mp9.The sequencing of the DNA was carried out by means of the method oftermination with dideoxyribonucleotides (Sanger, F. et al. (1977) Proc.Natl. Acad. Sci. USA, 74, 5463-5467).

2) Results

Analysis of the In Vitro Translation of mRNA Prepared from the SMG ofRats and Mice.

5 μg of total RNA obtained from the SMG of male and female mice or maleand female rats were translated in a cell-free system of reticulocytesin the presence of ³⁵S-methionine. The products of in vitro translationwere subjected to electrophoresis on a 12.5% sodiumdodecylsulfate-polyacrylamide gel and autoradiographed.

The comparison of the products of the in vitro translation of RNAprepared from the SMG of male and female rats shows the presence ofseveral polypeptides (of apparent molecular weights of 18,000, 19,000,35,000, 46,000) in larger amounts in the male rats than in the females.These data show the existence of a sexual dimorphism in the SMG of therat.

Furthermore, the comparison of these products of translation with thoseobtained with RNAs derived from SMG of mice shows that most of the majorpolypeptides are different in the two species. In particular, whereassex-linked differences could also be observed in the mouse, these latterdid not relate to the same polypeptides as in the rat. Conversely, theproducts of translation observed in the rat to be specific for the maleseem to be absent in the mouse.

Isolation and Sequence of a cDNA Complementary to a Specific mRNA of theMale Rat

In order to isolate mRNAs specific for the male from the SMG of the rat,a bank of cDNA prepared from this tissue was constructed in pUC9. Therecombinant clones were screened by utilizing a differential screeningstrategy such as that described in the Materials and Methods section.The positive clones were characterized by experiments of inhibition oftranslation of the mRNAs in a cell system by DNA-mRNA hybridization. Onecategory of recombinant cDNAs suppressed the in vitro synthesis of apolypeptide having an apparent molecular weight of 19,000 daltons. Thispolypeptide, designated SMR1, is present in the products of in vitrotranslation of RNAs from male rats but is not present in those fromfemales. The corresponding cDNA was utilized as probe in RNAhybridization experiments with transfer to a nitrocellulose membrane.For the hybridization a solution was used containing:

-   -   0.5 M sodium phosphate, pH 7.2    -   7% sodium dodecylsulfate (SDS)    -   1 mM EDTA    -   1% bovine serum albumin    -   sonicated salmon sperm DNA: 100 mg/ml

The experiment was done at 65° C. for 16 to 20 h.

4 washings of 10 minutes each were carried out at 65° C. with a solutioncontaining:

-   -   40 mM sodium phosphate, pH 7.2        -   1% SDS        -   1 mM EDTA

The cDNA is hybridized with a mRNA 700 nucleotides long, present inlarge amounts in the SMG of male rats. This inserted element of cDNA hasbeen characterized in more detail.

The sequence of the cDNA coding for SMR1 and the sequence deduced forthe protein are represented below

The inserted cDNA has a length of 652 nucleotides, if the poly(A)fragment is excluded. This sequence comprises an open reading frame of510 nucleotides. The only ATG which can serve as initiation codon issituated 73 nucleotides downstream from the 5′ end of the clone of cDNA.The untranslated region at the 3′ end (142 nucleotides long) containsthe consensus signal for polyadenylation (AATAAA), 23 nucleotidesupstream from the poly(A) tail (nucleotides 625-630). The translationstop codon is found in another AATAAA sequence (512-517) whichapparently is not recognized as polyadenylation signal.

The corresponding protein has a length of 146 amino acids, whichcorresponds to a molecular weight of about 16,000 daltons. Thismolecular weight is slightly lower than that determined from theelectrophoretic mobility of the in vitro translation product (19,000daltons). Such a difference between the molecular weight calculated fromthe analysis of the sequence and that determined from theelectrophoretic mobility has already been described for other proteinsof the SMG of the rat or the mouse.

SMR1 has a relatively high content of glutamine and proline residues,but does not contain a repetitive region. Hence, it does not belong tothe family of polypeptides “rich in proline” or “rich in glutamine”,which are essential proteins of the SMG. Furthermore, the sequence ofthe mRNA does not contain at its 5′ end the sequence of 80nucleotideswhich is characteristic of this family. However, like these proteins,SMR1 does not contain cystein or methionine residues (except in itssignal peptide).

The amino-terminal part of SMR1 is strongly hydrophobic, which ischaracteristic of signal peptides of most of the secreted proteins.Although the amino-terminal sequence of the mature protein has not beendetermined directly, from the statistical analysis made according to G.Von Heijne (Nucleic Acids Res. 14, 4683-4690, 1986) (rule “−3,−1”), itmay be supposed that the cleavage site of the signal peptide is locatedbetween the residues 18 and 19.

The protein SMR1 also shows certain features characteristic of theglycoproteins. The presence of two potential glycosylation sites linkedto N are observed at the positions 139 and 136. The protein isrelatively rich in proline (12%), threonine (12%) and glutamine (9.5%).Several glycosylation sites linked to 0 might thus be present in thecarboxy-terminal fragment of SMR1, since regions rich in proline andthreonine residues are usually present in highly O-glycosylatedproteins, such as the mucoproteins and the sialoglyco-proteins.

An interesting characteristic is the presence of pairs of basic aminoacids Arg-Arg at positions 27-28 and 33-34. Such dipeptides representpotential sites of cleavage by maturation enzymes (Lazure, C., et al(1983) Can. J. Biochem., Cell Biol. 61, 501-515 and Docherty, K. et al(1982) Ann. Rev. Physiol. 44, 625-638). They flank a tetrapeptideGln-His-Asn-Pro. The tetrapeptide and its adjacent, sequences arelocated in a hydrophilic environment which renders this regionaccessible to possible maturation enzymes.

The cleavage of Arg-Arg linkages by a maturation enzyme followed by theremoval of the basic residues by carboxypeptidase E (Fricker, L. D. etal (1983) J. Biol. Chem. 258, 10950-10955) and possibly anaminopeptidase (Loh, Y. P. et al. (1984) Ann. Rev. Neurosci. 7, 189-222)would produce a mixture of tetrapeptide (Gln-His-Asn-Pro) andpentapeptide (Gln-His-Asn-Pro-Arg), since “Pro-Arg” is not a goodsubstrate for carboxypeptidase E. Other post-translational modificationscould also include the formation of pyroGlu acid derivatives of theseproducts, giving rise to a mixture of pyroGlu-His-Asn-Pro-Arg andpyroGlu-His-Asn-Pro. These structures recall those of thyroliberin(TRH).

Regulation of the Accumulation of mRNA Coding for SMR1 in the SMG of theRat by Androgens.

In order to study the regulation of the accumulation of mRNA coding forSMR1 in the SMG of the rat by androgens, mRNA containing a poly(A)sequence was prepared from the SMG of adult males, males castrated 20days previously, castrated males subjected to a treatment withandrogens, females and females treated with androgens. 1 μg of total RNAfrom male rats, female rats treated with testosterone, castrated malerats, castrated male rats treated with testosterone and female rats wassubjected to electrophoresis in a 1.4% agarose-formaldehyde gel, theseRNAs were transferred to a Nylon membrane and were hybridized with thecDNA probe coding for SMR1. The time of exposure for autoradiography was2 hours. The results of the RNA analysis, by transfer of these mRNAs tosolid supports, by means of a SMR1 probe labelled with ³²P are shown inthe figure (part A). A considerable difference in the accumulation ofmRNA coding for SMR1 is observed in the SMG of male rats and those offemales.

Furthermore, various amounts (as indicated in the figure) of RNA fromthe SMG of male rats and female rats were subjected to electrophoresison a 2% agarose-formaldehyde gel, they were transferred to filters andhybridized with the cDNA probe coding for SMR1. The film was exposed for30 hours. As is apparent in part B, the mRNA coding for SMR1 accumulatesin very large quantities in the SMG of male Wistar rats, since an amountas low as 1.5 ng of total RNA was sufficient to give a hybridizationsignal. Conversely, the level of accumulation of mRNA coding for SMR1 inthe SMG of female Wistar rats was about 1,000 to 3,000 times lower thanthat in males.

In the castrated males, the quantity of mRNA coding for SMR1 was reduced10 to 20 fold. The administration of testosterone to these malesrestored the amount of mRNA coding for SMR1 to the same value as thatobserved in the non-castrated males. Furthermore, the administration oftestosterone to adult female rats caused the accumulation of mRNA codingfor SMR1 in amounts similar to that observed in the males.

A remarkable property of the mRNA coding for SMR1 is its high degree ofaccumulation in the SMG of the rat in response to a treatment withandrogens. This strongly suggests that SMR1 is synthesized in the GCTcells of the SMG (just like the EGF, NGF and renin as well as otherproteins under the control of androgens in the SMG of the mouse).Furthermore, the difference in the level of accumulation of mRNA codingfor SMR1 in the male and in the female is very great (greater than threeorders of magnitude), in comparison with that usually observed for othergenes controlled by antigens in the target organs (kidney, liver, SMG).

These results, and in particular the high degree of induction of theSMR1 gene by androgens, suggest that SMR1 may fulfil an importantfunction specific for the male in the rat. SMR1 might be the precursorof a molecule (the tetra- or pentapeptides or the C-terminal part ofSMR1) controlling behavioural characteristics in the male rat.

The products described in the present invention can be used fortherapeutic purposes or as laboratory reagents.

1-14. (canceled)
 15. An isolated nucleic acid having the nucleotidesequence of nucleotides 1 to 510 of SEQ ID NO: 7, or a sequencehybridizing thereto, in a hybridization solution containing: 0.75 Msodium phosphate, pH 7.2, 7% sodium dodecylsulfate, 1 mM EDTA, 1% bovineserum albumin, and sonicated salmon sperm DNA: 100 mg/ml, at 65° C.; andafter washing four times for 10 minutes at 65° C. with a solutioncontaining: 40 mM sodium phosphate, pH 7.2, 1% sodium dodccylsulfate,and 1 mM EDTA.